The present invention relates to methods of identifying compounds capable of regulating the activity of mutant enzymes, to computing platforms capable of generating models representing 3D structures of crystallized enzymes, to crystallized enzymes, and to methods of crystallizing enzymes. In particular, embodiments of the present invention relate to methods of identifying compounds capable of correcting impaired enzymatic activity of mutant human glucocerebrosidase molecules associated with Gaucher disease, to computing platforms capable of generating models representing essentially complete experimentally determined 3D structures of crystallized human glucocerebrosidase polypeptide, to crystallized human glucocerebrosidase polypeptide, and to methods of crystallizing human glucocerebrosidase polypeptide. The present invention further relates to preparations of partially glycosylated glucocerebrosidase molecules having about the same capacity to catalyze hydrolysis of a glucocerebroside as preparations of fully glycosylated glucocerebrosidase molecules, such as Cerezyme®, having optimal stability of such capacity under physiological conditions, and being capable of undergoing significant internalization/uptake by a phagocyte, such as a macrophage. In particular, the present invention relates to methods of using such preparations for treating diseases associated with glucocerebrosidase deficiency, such as Gaucher disease.
Gaucher disease, the most common lysosomal storage disease (Meikle P J. et al., 1999. JAMA 281:249-54), is a highly debilitating disease occurring with a frequency of 1 in 40,000-60,000 in the general population, and 1 in 500-1,000 amongst Ashkenazi Jews [Beutler E. and Grabowski G A., in: “The Metabolic and Molecular Bases of Inherited Disease”, Scriver C R. et al. (eds.), McGraw-Hill Inc., pp. 3635-3668 (2001)]. Due to its genetic component, Gaucher disease represents a genetic testing dilemma for potential carriers. The disease occurs in various forms, in particular Type 1 which is predominantly characterized by hepatosplenomegaly; and Types 2 and 3 which are characterized by early or chronic onset of severe neurological symptoms. In Gaucher disease, deficiency in enzymatic activity of the lysosomal enzyme glucocerebrosidase due to mutations in the enzyme [Beutler E. and Grabowski G A., in: “The Metabolic and Molecular Bases of Inherited Disease”, Scriver C R. et al. (eds.), McGraw-Hill Inc., pp. 3635-3668 (2001)] leads to pathological lysosomal accumulation of the lipid glucosylceramide (GlcCer or glucocerebroside), principally in macrophages, but also in monocytes, and, in severe cases, in neurons. The lysosomal accumulation of glucosylceramide leads to buildup of congested lysosomes at various anatomic sites throughout the body, including the liver, spleen and bone marrow, which in turn causes anemia and osteopenia. Gaucher disease may also be associated with highly debilitating conditions, such as glomerulonephritis, pericarditis, pericardial calcification, haemorrhagic colitis and/or amyloidosis.
Glucosylceramide, whose pathological accumulation is associated with Gaucher disease, belongs to a family of lipids called sphingolipids which play important roles in normal cell physiology. Gaucher disease is one of the group of “sphingolipid storage diseases” which also include Tay-Sachs and Niemann-Pick disease. Sphingolipids have received huge attention over the past couple of decades since it was shown that they are involved in regulation of cell physiology via signal transduction. While thousands of scientific papers have been published during this period on basic aspects of sphingolipid research, surprisingly little is known about the molecular mechanisms by which sphingolipid accumulation leads to sphingolipid storage diseases, or in the case of Gaucher disease, how glucosylceramide accumulation causes cell dysfunction. In 1999, the present inventors performed an experiment which gave clues to the pathophysiological mechanism that might be responsible for neurological disease in types 2 and 3 Gaucher disease. Over the past 4 to 5 years, these findings have been formulated into the “calcium hypothesis” in which the present inventors suggested that defective calcium homeostasis is responsible for the altered neuronal dysfunction in neuronopathic forms of Gaucher disease, which results in brain dysfunction (work summarized in: Korkotian, E. et al., 1999. J. Biol. Chem. 274:21673-21678; Pelled, D. et al., 2000. J. Inh. Met. Dis. 23, 175-184; Lloyd-Evans, E. et al., 2003. J. Biol. Chem. 278:23594-23599; Pelled, D. et al., Enhanced calcium release in the acute neuronopathic form of Gaucher disease. Submitted for publication).
The enzyme glucocerebrosidase (EC 3.2.1.45, acid beta-glucosidase, D-glucosyl-N-acylsphingosine glucohydrolase, glucosylceramidase) is a peripheral membrane protein which hydrolyzes the beta-glucosyl linkage of glucosylceramide in lysosomes, thereby generating beta-glucose and ceramide (FIG. 1a). This enzymatic activity requires the coordinate action of saposin C and negatively-charged lipids for maximal activity [Beutler E. and Grabowski G A., in: “The Metabolic and Molecular Bases of Inherited Disease”, Scriver C R. et al. (eds.), McGraw-Hill Inc., pp. 3635-3668 (2001); Grabowski G A. et al., 1990. Critical Rev Biochem Mol Biol. 25:385-414]. Based on sequence similarity, glucocerebrosidase is classified as a member of glycoside hydrolase family 30 (see website of ARCHITECTURE ET FONCTION DES MACROMOLECULES BIOLOGIQUES, France).
Of the approximately 200 known glucocerebrosidase mutations, homozygosity for the common mutations Asn370Ser and Leu444Pro is associated with non-neuronopathic (Charrow J. et al., 2000. Arch Intern Med. 160:2835-43) and neuronopathic [Erikson A. et al. in: “Gaucher Disease”, Zimran A. (ed.), Bailliere Tindall, London, pp. 711-723 (1997)] Gaucher disease, respectively. Mutation Asn370Ser is the most common mutation, accounting for about 70 and 25 percent of the mutant alleles in Ashkenazi Jewish and non-Jewish patients, respectively [Beutler E. and Grabowski G A., in: “The Metabolic and Molecular Bases of Inherited Disease”, Scriver C R. et al. (eds.), McGraw-Hill Inc., pp. 3635-3668 (2001)]. Many of the glucocerebrosidase mutations (FIG. 1d) are rare and restricted to a few individuals, and most partially or entirely decrease catalytic activity (Meivar-Levy, I. et al., 1994. Biochem. J. 303:377-382) or may reduce glucocerebrosidase stability (Grace M E. et al., 1994. J Biol. Chem. 269:2283-2291).
Whole-enzyme replacement therapy with Cerezyme®, a recombinant variant of human glucocerebrosidase (Grabowski G A. et al., 1995. Ann Intern Med. 122:33-9) is the main treatment for Type 1 Gaucher disease. Such treatment, however, is not curative, nor does it satisfactorily alleviate the symptoms of the disease. Furthermore, such whole-enzyme replacement therapy has numerous significant disadvantages, including: (i) administration of a molecule having various suboptimal pharmacokinetic characteristics, including suboptimal tissue penetration as a result of its large size; and suboptimal plasma membrane permeability and in-vivo half-life due to its polypeptidic composition; (ii) incapacity to correct endogenous glucocerebrosidase enzyme activity, and thereby incapacity to therapeutically confer such activity with optimal spatial (cell type/subcellular location), temporal, and activity level regulation; (iii) for optimal therapeutic results, the need to administer the enzyme via injection, a painful, inconvenient, and expensive process; and (iv) elicitation of harmful immune responses against the administered enzyme in a substantial proportion of treated subjects as a result of its polypeptidic/modified oligosaccharide chemical composition (see entry for “Cerezyme” in the website of the Gaucher Registry. Hence, there is a clearly felt need for novel/improved Gaucher disease drugs.
One approach for attempting to correct the defective enzymatic activity of the glucocerebrosidase Asn370Ser mutant which has been proposed in the prior art involves utilizing deoxynijirimycin (DNJ)-based compounds (Sawkar et al., 2002. Proc Natl Acad Sci USA. 99:15428-33). Such an approach, however, suffers from significant drawbacks, including: (i) the nonspecific inhibitory effect of DNJ compounds on enzymes that make and break glucosyl bonds, such as endoplasmic reticulum oligosaccharide-processing enzymes alpha-glucosidase I/II, ceramide glucosyl transferase, and both non-lysosomal and lysosomal glucocerebrosidase; (ii) the significant toxicity displayed by DNJ compounds; and (iii) the fact that DNJ compounds have not been demonstrated to have a significant restorative effect on the impaired enzymatic activity of any glucocerebrosidase mutant other than Asn370Ser, including a demonstrated ineffectiveness for correcting impaired enzymatic activity of the common Leu444Pro glucocerebrosidase mutant.
Thus, in sharp contrast to prior art Gaucher disease drugs, optimal Gaucher disease drugs, would be compounds having optimally small dimensions, a non-polypeptidic chemical composition, a capacity to correct impaired enzymatic activity of any of various glucocerebrosidase mutants, and would be capable of correcting the impaired enzymatic activity of glucocerebrosidase mutants associated with Gaucher disease in-vivo with optimal effectiveness and safety.
Ideally, such compounds could be computationally identified by obtaining sets of structure coordinates defining experimentally determined 3D structures of glucocerebrosidase at atomic resolution, using such sets of structure coordinates for producing computational platforms capable of generating models representing such 3D structures of human glucocerebrosidase at such atomic resolution, and using such computing platforms for computationally identifying compounds capable of interacting with mutant human glucocerebrosidase molecules associated with Gaucher disease in such a way as to correct impaired enzymatic activity thereof.
One prior art approach which has been employed involves using two-dimensional hydrophobic cluster analysis in attempts to provide sets of structure coordinates defining predictive structures of human glucocerebrosidase molecules (Fabrega S. et al., 2002. J Soc Biol. 196:151-60; Fabrega S. et al., 2000. Glycobiology 10:1217-24).
Another approach which has been employed involves crystallizing Cerezyme® and analyzing such crystals via X-ray crystallography in attempts to generate X-ray diffraction data defining 3D structures of Cerezyme® suitable for computational identification of novel Gaucher disease drugs (Roeber D. et al., 2003. Acta Cryst. D59:343-344).
These prior art approaches, however, have essentially failed. Approaches involving predictive methods based on two-dimensional hydrophobic cluster analysis are suboptimal due to the significant inaccuracies inherent to such predictive methods, and, in any case, have not provided sets of structure coordinates defining the structure of glucocerebrosidase, nor of significant portions thereof, at adequately high resolution, with satisfactory completeness, and with a satisfactory degree of accuracy. Furthermore, approaches involving crystallization of Cerezyme® have not succeeded in producing crystals capable of generating X-ray diffraction data defining structures of Cerezyme® or portions thereof.
Various partially glycosylated glucocerebrosidase preparations have been described in the prior art (as used herein, the phrase “partially glycosylated glucocerebrosidase” refers to a glucocerebrosidase molecule whose amino acid sequence includes at least one fully unglycosylated amino acid residue at a position normally corresponding to a glycosylated Asn residue).
One such approach involves individually or collectively replacing in human glucocerebrosidase via site-directed mutagenesis Asn residues which are glycosylated with a non-glycosylated amino acid residue (Berg-Fussman A. et al., 1993. J Biol Chem. 268:14861-14866) to generate, for each amino acid residue at a position corresponding a normally glycosylated Asn residue, a point-mutant glucocerebrosidase preparation having one unglycosylated amino acid residue at a position corresponding a normally glycosylated Asn residue, or a quadruple point-mutant glucocerebrosidase preparation having all four amino acid residues at positions corresponding a normally glycosylated Asn residue such residues being unglycosylated.
Another approach involves subjecting porcine glucocerebrosidase to sequential deglycosylation with endoglycosidase H so as to obtain preparations of porcine glucocerebrosidase having only three, only two or only one glycosylated Asn residues out of the four normally glycosylated Asn residues (Erickson A H et al., 1985. J Biol Chem. 260:14319-24).
These prior art partially glycosylated glucocerebrosidase preparations are suboptimal for treatment of a disease associated with glucocerebrosidase deficiency, such as Gaucher disease in humans, via enzyme replacement therapy for various reasons. The approach involving mutagenesis failed to generate a mutant glucocerebrosidase preparation demonstrably having either improved enzymatic activity, or improved stability of such activity under physiological conditions. The approach employing enzymatic deglycosylation failed to demonstrate that any of the glycoforms generated has any enzymatic activity, failed to characterize which Asn residues were glycosylated/deglycosylated in any of the preparations, and involve a glucocerebrosidase of porcine origin.
Thus, all prior art approaches have failed to provide an adequate solution for producing computational platforms suitable for computationally identifying compounds capable of interacting with mutant human glucocerebrosidase molecules associated with Gaucher disease in such a way as to correct impaired enzymatic activity thereof, and have failed to provide an adequate solution for treatment of Gaucher disease via enzyme replacement therapy.
There is thus a widely recognized need for, and it would be highly advantageous to have, a computing platform for identifying optimal Gaucher disease drugs, and a glucocerebrosidase preparation for treating Gaucher disease devoid of the above limitations.